Human Ovary Research Articles

Unraveling the mechanisms of chemotherapy-induced damage to human primordial follicle reserve: road to developing therapeutics for fertility preservation and reversing ovarian aging.

Among the investigated mechanisms of chemotherapy-induced damage to human primordial follicle reserve are induction of DNA double-strand breaks (DSBs) and resultant apoptotic death, stromal-microvascular damage and follicle activation. Accumulating basic and translational evidence suggests that acute exposure to gonadotoxic chemotherapeutics, such as cyclophosphamide or doxorubicin, induces DNA DSBs and triggers apoptotic death of primordial follicle oocytes within 12-24 h, resulting in the massive loss of ovarian reserve. Evidence also indicates that chemotherapeutic agents can cause microvascular and stromal damage, induce hypoxia and indirectly affect ovarian reserve. While it is possible that the acute reduction of the primordial follicle reserve by massive apoptotic losses may result in delayed activation of some primordial follicles, this is unlikely to be a predominant mechanism of loss in humans. Here, we review these mechanisms of chemotherapy-induced ovarian reserve depletion and the potential reasons for the discrepancies among the studies. Based on the current literature, we propose an integrated hypothesis that explains both the acute and delayed chemotherapy-induced loss of primordial follicle reserve in the human ovary.

Homozygous hypomorphic BRCA2 variant in primary ovarian insufficiency without cancer or Fanconi anaemia trait

BackgroundPrimary ovarian insufficiency (POI) affects 1% of women under 40 years and is a public health problem. The genetic causes of POI are highly heterogeneous with isolated or syndromic forms....

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

PTEN and FOXO3 expression in the prenatal and postnatal human ovary

The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

CCL20-CCR6 axis directs sperm-oocyte interaction and its dysregulation correlates/associates with male infertility‡.

The interaction of sperm with the oocyte is pivotal during the process of mammalian fertilization. The limited numbers of sperm that reach the fallopian tube as well as anatomic restrictions indicate that human sperm-oocyte encounter is not a matter of chance but a directed process. Chemotaxis is the proposed mechanism for re-orientating sperm toward the source of a chemoattractant and hence to the oocyte. Chemokines represent a superfamily of small (8-11kDa), cytokine-like proteins that have been shown to mediate chemotaxis and tissue-specific homing of leukocytes through binding to specific chemokine receptors such as CCRs. Here we show that CCR6 is abundantly expressed on human sperms and in human testes. Furthermore, radioligand-binding experiments showed that CCL20 bound human sperm in a specific manner. Conversely, granulosa cells of the oocyte-surrounding cumulus complex as well as human oocytes represent an abundant source of the CCR6-specific ligand CCL20. In human ovaries, CCL20 shows a cycle-dependent expression pattern with peak expression in the preovulatory phase and CCL20 protein induces chemotactic responses of human sperm. Neutralization of CCL20 in ovarian follicular fluid significantly impairs sperm migratory responses. Conversely, analyses in infertile men with inflammatory conditions of the reproductive organs demonstrate a significant increase of CCL20/CCR6 expression in testis and ejaculate. Taken together, findings of the present study suggest that CCR6-CCL20 interaction may represent an important factor in directing sperm-oocyte interaction.

Maternally contributed Nlrp9b expressed in human and mouse ovarian follicles contributes to early murine preimplantation development.

The aim of the study is to investigate presence and role of the gene encoding the maternally contributed nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) in human and mouse ovaries, respectively, and in preimplantation mouse embryo development by knocking down Nlrp9b. Expression levels of NLRP9 mRNA in human follicles were extracted from RNA sequencing data from previous studies. In this study, we performed a qPCR analysis of Nlpr9b mRNA in mouse oocytes and found it present. Intracellular ovarian distribution of NLRP9B protein was accomplished using immunohistochemistry. The distribution of NLRP9B was explored using a reporter gene approach, fusing NLRP9B to green fluorescent protein and microinjection of in vitro-generated mRNA. Nlrp9b mRNA function was knocked down by microinjection of short interference (si) RNA targeting Nlrp9b, into mouse pronuclear zygotes. Knockdown of the Nlrp9b mRNA transcript was confirmed by qPCR. We found that the human NLRP9 gene and its corresponding protein are highly expressed in human primordial and primary follicles. The NLRP9B protein is localized to the cytoplasm in the blastomeres of a 2-cell embryo in mice. SiRNA-mediated knockdown of Nlrp9b caused rapid elimination of endogenous Nlrp9b mRNA and premature embryo arrest at the 2- to 4-cell stages compared with that of the siRNA-scrambled control group. These results suggest that mouse Nlrp9b, as a maternal effect gene, could contribute to mouse preimplantation embryo development. It remains to investigate whether NLRP9 have a crucial role in human preimplantation embryo and infertility.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines. Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode. This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ methodgenerates reliable results and can be further applied to other in vitro cellular models.

OR31-03 Single-Cell Profiling of Adult Human Ovarian Cortex Reveals Six Main Cell Types but No Germline Stem Cells

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been suggested that oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Our study aimed at comprehensive characterization of all cell types present in the ovarian cortex, including the previously reported oogonial stem cells. We developed methods to dissociate human ovarian cortex to a viable single cell solution allowing subsequent analysis by single cell transcriptomic profiling and cell surface antigen screening. In all analyses, cells captured by DDX4 antibodies (DDX4 Ab+) were included as a reference. High quality ovarian cortex tissue from gender reassignement and caesarean section patients was used in the analyses. Our single cell transcriptomic analyses based on >24,000 cells revealed the presence of six main cell types in ovarian cortex; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Surface marker screening showed robust expression of 43 cell surface antigens in ovarian cortex cells. With the help of transcriptomic and cell surface antigen profiles, the DDX4 Ab+ cells were identified as perivascular cells. This finding was validated by immunostaining of ovarian tissue showing DDX4 Ab+ cells lining CD31 positive endothelial cells of blood vessels. To search for germline stem cells on a broader front, we compared our data with human fetal ovary cells including pre-meiotic germ cells (Li et al. 2017) and found no evidence for the presence of germ line stem cells of any kind in adult human ovarian cortex. In summary, we provide the first cell map of human ovarian cortex. Our results demonstrate six main cell types, but cannot provide support to the existence of oogonial stem cells. This dataset will be a valuable tool for studying the role of specific cell populations in ovarian biology, dissecting causes of infertility, and developing novel assisted reproductive technologies or even contraceptives.

OR24-04 Ovarian Follicle Survival Is Determined by Follicle-Stimulating Hormone Receptor (FSHR) and Estrogen Receptor (GPER) Heteromers

Mechanisms regulating the selection of antral ovarian follicles are poorly understood and supposed to rely on low estrogen levels, decline of follicle-stimulating hormone (FSH) levels and receptor (FSHR) expression. These concepts are challenged in vitro, where apoptosis of human granulosa cells (hGLC) and transfected cell lines is induced by high doses of FSH or FSHR overexpression, while estrogens induce anti-apoptotic signals via nuclears and a G protein-coupled estrogen receptor (GPER). Therefore, in vitro data suggest that antral follicle selection may be driven by underestimated, FSH/FSHR-dependent apoptotic signals due to transiently maximized FSHR expression and overload of cAMP signalling, prevailing on estrogen-dependent signals. Here we demonstrate how FSHR/GPER physical interaction rescue ovarian follicles from FSH-mediated death. 10 nM FSH induces high intracellular levels of cAMP, measured by bioluminescence resonance energy transfer (BRET), and apoptosis in cultured hGLC under conditions where GPER levels are depleted by siRNA. This result was confirmed in transfected HEK293 cells overexpressing FSHR. Using BRET, photo-activated localization microscopy (PALM) and bioinformatics prodiction, we also demonstrate FSHR/GPER heteromers at the cell surface. The role of FSHR/GPER heteromers may be relevant to inhibit FSH-induced death signals, since increasing GPER expression levels in HEK293 cells co-expressing FSHR results in displacement of the Gαs-protein to FSHR, blockade of FSH-induced cAMP production and inhibition of apoptosis. However, in HEK293 cells coexpressing GPER/FSHR, FSH-induced activation of the anti-apoptotic AKT-pathway via a Gβγ-dependent mechanism, as demonstrated by Western blotting in cells treated using the inhibitor gallein. Inhibition of both FSH-induced cAMP production and apoptosis was lost when FSHR is coexpressed together with a mutant GPER, unable to heteromerize with FSHR, as well as in KO HEK293 cells unable to produce a molecular complex associated with GPER inhibiting cAMP. GPER/FSHR coexpression is confirmed in secondary follicles from paraffin-embedded tissues of human ovary by immunohistochemistry, suggesting that FSHR-GPER heterodimers could be physiologically relevant in vivo for inhibiting cAMP-linked apoptosis. Most importantly, FSHR and GPER co-expression correlates in hGLC from FSH-normo-responder women undergoing assisted reproduction, while it is not in hGLC from FSH-poor-responders, where increasing FSHR mRNA levels do not correspond to increasing GPER mRNA levels. We demonstrate that death signals in atretic follicles are delivered through overexpressed FSHR and inhibited by FSHR/GPER heteromerization, activating anti-apoptotic pathways. This finding unveils a novel working model of the physiology of dominant follicle selection and the relationship between FSH and estrogens.

Dynamics of the transcriptional landscape during human fetal testis and ovary development.

Which transcriptional program triggers sex differentiation in bipotential gonads and downstream cellular events governing fetal testis and ovary development in humans? The characterization of a dynamically regulated protein-coding and non-coding transcriptional landscape in developing human gonads of both sexes highlights a large number of potential key regulators that show an early sexually dimorphic expression pattern. Gonadal sex differentiation is orchestrated by a sexually dimorphic gene expression program in XX and XY developing fetal gonads. A comprehensive characterization of its non-coding counterpart offers promising perspectives for deciphering the molecular events underpinning gonad development and for a complete understanding of the etiology of disorders of sex development in humans. To further investigate the protein-coding and non-coding transcriptional landscape during gonad differentiation, we used RNA-sequencing (RNA-seq) and characterized the RNA content of human fetal testis (N = 24) and ovaries (N = 24) from 6 to 17 postconceptional week (PCW), a key period in sex determination and gonad development. First trimester fetuses (6-12 PCW) and second trimester fetuses (13-14 and 17 PCW) were obtained from legally induced normally progressing terminations of pregnancy. Total RNA was extracted from whole human fetal gonads and sequenced as paired-end 2 × 50 base reads. Resulting sequences were mapped to the human genome, allowing for the assembly and quantification of corresponding transcripts. This RNA-seq analysis of human fetal testes and ovaries at seven key developmental stages led to the reconstruction of 22 080 transcripts differentially expressed during testicular and/or ovarian development. In addition to 8935 transcripts displaying sex-independent differential expression during gonad development, the comparison of testes and ovaries enabled the discrimination of 13 145 transcripts that show a sexually dimorphic expression profile. The latter include 1479 transcripts differentially expressed as early as 6 PCW, including 39 transcription factors, 40 long non-coding RNAs and 20 novel genes. Despite the use of stringent filtration criteria (expression cut-off of at least 1 fragment per kilobase of exon model per million reads mapped, fold change of at least 2 and false discovery rate adjusted P values of less than <1%), the possibility of assembly artifacts and of false-positive differentially expressed transcripts cannot be fully ruled out. Raw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI GEO under accession number GSE116278. The intrinsic nature of this bulk analysis, i.e. the sequencing of transcripts from whole gonads, does not allow direct identification of the cellular origin(s) of the transcripts characterized. Potential cellular dilution effects (e.g. as a result of distinct proliferation rates in XX and XY gonads) may account for a few of the expression profiles identified as being sexually dimorphic. Finally, transcriptome alterations that would result from exposure to pre-abortive drugs cannot be completely excluded. Although we demonstrated the high quality of the sorted cell populations used for experimental validations using quantitative RT-PCR, it cannot be totally excluded that some germline expression may correspond to cell contamination by, for example, macrophages. For the first time, this study has led to the identification of 1000 protein-coding and non-coding candidate genes showing an early, sexually dimorphic, expression pattern that have not previously been associated with sex differentiation. Collectively, these results increase our understanding of gonad development in humans, and contribute significantly to the identification of new candidate genes involved in fetal gonad differentiation. The results also provide a unique resource that may improve our understanding of the fetal origin of testicular and ovarian dysgenesis syndromes, including cryptorchidism and testicular cancers. This work was supported by the French National Institute of Health and Medical Research (Inserm), the University of Rennes 1, the French School of Public Health (EHESP), the Swiss National Science Foundation [SNF n° CRS115_171007 to B.J.], the French National Research Agency [ANR n° 16-CE14-0017-02 and n° 18-CE14-0038-02 to F.C.], the Medical Research Council [MR/L010011/1 to P.A.F.] and the European Community's Seventh Framework Programme (FP7/2007-2013) [under grant agreement no 212885 to P.A.F.] and from the European Union's Horizon 2020 Research and Innovation Programme [under grant agreement no 825100 to P.A.F. and S.M.G.]. There are no competing interests related to this study.

R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth

R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/β-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility.

Prior and novel coronaviruses, Coronavirus Disease 2019 (COVID-19), and human reproduction: what is known?

ObjectiveTo summarize current understanding of the effects of novel and prior coronaviruses on human reproduction, specifically male and female gametes, and in pregnancy.DesignReview of English publications in PubMed and Embase to April 6, 2020.Method(s)Articles were screened for reports including coronavirus, reproduction, pathophysiology, and pregnancy.Intervention(s)None.Main Outcome Measure(s)Reproductive outcomes, effects on gametes, pregnancy outcomes, and neonatal complications.Result(s)Seventy-nine reports formed the basis of the review. Coronavirus binding to cells involves the S1 domain of the spike protein to receptors present in reproductive tissues, including angiotensin-converting enzyme-2 (ACE2), CD26, Ezrin, and cyclophilins. Severe Acute Respiratory Syndrome Coronavirus 1 (SARS–CoV-1) may cause severe orchitis leading to germ cell destruction in males. Reports indicate decreased sperm concentration and motility for 72–90 days following Coronavirus Disease 2019 (COVID-19) infection. Gonadotropin-dependent expression of ACE2 was found in human ovaries, but it is unclear whether SARS–Coronavirus 2 (CoV-2) adversely affects female gametogenesis. Evidence suggests that COVID-19 infection has a lower maternal case fatality rate than SARS or Middle East respiratory syndrome (MERS), but anecdotal reports suggest that infected, asymptomatic women may develop respiratory symptoms postpartum. Coronavirus Disease 2019 infections in pregnancy are associated with preterm delivery. Postpartum neonatal transmission from mother to child has been reported.Conclusion(s)Coronavirus Disease 2019 infection may affect adversely some pregnant women and their offspring. Additional studies are needed to assess effects of SARS–CoV-2 infection on male and female fertility.

Unraveling the metabolic and machinery constraints on protein secretion through a novel systems biology framework

In mammalian cells, metabolism is a core process driving homeostasis, but variations in other cell processes largely define cell type identity and cell‐type specific functions. The profile of secreted and membrane proteins show substantial cell‐type specificity and drive many tissue specific functions. These proteins, encoded by up to 1/3 of mammalian protein‐coding genes, include hormones, membrane proteins, and extracellular enzymes, and these are synthesized and trafficked through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling its impact on diverse proteins is particularly important since the pathway is implicated in many diseases and harnessed for biopharmaceutical production. Through the use of network reconstruction approaches and protein interaction assays, we have mapped out the core secretory pathway and integrated it with our genome‐scale metabolic models of human, mouse, and Chinese hamster ovary cells. We first deploy graph‐based approaches to evaluate the dependency of protein secretion on diverse human secreted proteins. We then deployed constraint‐based modeling to quantify the bioenergetic demands for the synthesis and secretion of these proteins. Finally, we deployed these models to engineer mammalian cells for enhanced secretion of high‐value biologic drugs. Thus, we present a platform that enables the study and engineering of the mammalian secretory pathway and metabolism for systems biotechnology.Support or Funding InformationNIGMS R35 GM119850 and the Novo Nordisk Foundation Center for Biosustainability NNF10CC1016517

HIGHLY INCREASED VASCULAR DENSITY IN CORPORA LUTEA OF PD-L1 KNOCKOUT MICE COMPARED TO CONTROLS

Programmed cell death protein-1 (PD-1) and its ligands (PD-L1 and PD-L2) function within an important checkpoint for immune cell modulation. While expression of PD-1 and its ligands is mostly limited to lymphoid and myeloid tissue, our group has shown that both membrane-bound and soluble variants of PD-1, PD-L1, and PD-L2 are expressed in the human and murine ovary, including by granulosa cells, oocytes and cells of the corpus luteum. Because of recent studies showing that PD-L1 plays an inhibitory role in angiogenesis within the lymphatic vasculature (1) and the cornea (2), we hypothesized that deficiency for the ligand might impact the highly dynamic process of blood vessel development within corpora lutea, and possibly, numbers of immature follicles.

Phytochemical Screening and Cytotoxic Properties of Ethanolic Extract of Young and Mature Khat Leaves

The khat plant has been culturally used in many parts of Africa and the Arabian Peninsula for many years to induce psycho-stimulating effect. Because of the global wide-spreading nature, khat chewing is being considered as a universally growing problem. Catha abbottii, Catha edulis, and Catha transvaalensis are the three species of khat commonly chewed in Saudi Arabia and nearby regions. Khat users usually prefer to chew young leaves over mature ones due to the diverse effects produced by both. Though many of the constituents of khat leaves have been identified, the complete phytochemical profile of young and mature leaves was not performed or compared; also, no evidence is available to affirm the cytotoxicity of young or mature leaves. Therefore, this study aimed to investigate the phytochemical basis of the differential response of the young and mature leaves and to assess the cytotoxicity of young and mature khat leaves. Ethanolic extracts of young and mature leaves of three khat cultivars were subjected to GC-MS. Hierarchical cluster analysis revealed the existence of two major clusters. The extracts of young leaves were found to contain the maximum content of cathinone; however, methoxyamphetamine was found in only one extract of young leaves. Cytotoxicity investigations were also conducted on both types of leaves using three cancer cell lines, human breast adenocarcinoma, human ovary adenocarcinoma, and human colon adenocarcinoma and also normal human fetal lung fibroblast cell line was used. All extracts showed comparable cytotoxicity, IC50 ranging from 22–59 μg/mL on the cancer cells; however, we observed more cytotoxicity against normal cells (IC50: 6–41 μg/mL). The predominant cytotoxicity on normal cells may pose many health hazards to khat consumers.

A primate perspective on oocytes and transgenerational PCOS.

'Androgenized' rodent models are widely used to explore the pathophysiology underlying human polycystic ovary syndrome (PCOS), including reproductive and metabolic dysfunction. Based on a recent study using a dihydrotestosterone (DHT)-treated murine model, it has been proposed that prenatal androgen excess alone can predispose to transgenerational transmission of PCOS. From RNA sequencing analysis of metaphase II (MII) oocytes of androgenized lineages, the authors speculated that oocyte factors, including up-regulation of cytotoxic granulosa-associated RNA binding protein-like 1 (TiaL1), are sufficient to promote disease transfer across generations. Although this is an intriguing concept, it was not considered in the context of earlier publications in which the transcriptomes of human MII oocytes from PCOS women undergoing IVF were compared with women without PCOS. In one of these papers, a number of differentially expressed genes in PCOS MII oocytes (TIAL1 was not differentially expressed) were found to have putative response elements in their promoters for androgen receptors and peroxisome proliferating receptor gamma, providing a mechanism for how excess androgens and/or metabolic defects associated with PCOS might affect female germ cells.

Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.

Human BMP8A suppresses luteinization of rat granulosa cells via the SMAD1/5/8 pathway.

Bone morphogenetic proteins (BMPs) are known to play an indispensable role in preventing the precocious luteinization of granulosa cells within growing ovarian follicles. In this study, we found that the transcripts of BMP8 genes are enriched in the ovaries of humans and rodents. When analyzing transcriptomic datasets obtained from human mature granulosa cells, we further found that the BMP8 transcripts not only show the highest abundance among the searchable BMP-related ligands but also decrease significantly in women of advanced age or women with polycystic ovarian syndrome. The correlation between the BMP8 levels in granulosa cells and the decline in ovarian function in these subjects suggests that BMP8 protein may be involved in the regulation of granulosa cell function(s). Using a rat model, we demonstrated that human BMP8A protein activates the SMAD1/5/8 and the SMAD2/3 pathways simultaneously in both immature and mature granulosa cells. Furthermore, the expression of potential type I and type II receptors used by BMP8 in rat granulosa cells was characterized. We found that BMP8A treatment can significantly inhibit gonadotropin-induced progesterone production and steroidogenesis-related gene expression in granulosa cells. Pathway dissection using receptor inhibitors further revealed that such inhibitory effects occur specifically through the BMP8-activated SMAD1/5/8, but not SMAD2/3, pathway. Taken together, considering its abundance and possible functions in granulosa cells, we suggest that BMP8 may act as a novel luteinization inhibitor in growing follicles.

Effect of high-fat diet on the various morphological parameters of the ovary.

Increased food consumption rich in fat and carbohydrate and sedentary lifestyle have seriously increased the rates of obesity and obesity-associated diseases in developed countries. Female mice with diet-induced obesity exhibit infertility and thus can serve as a model for human polycystic ovary syndrome. The aim of the present study was to examine how ovary is affected by diet-induced obesity. The effects of high-fat diet (HFD) on ovary morphology in mice fed with HFD were investigated using unbiased stereological methods. The ovary of mice fed with HFD (n=8, C1090-60, Altromine) for 9 weeks, were compared with that of mice fed with standard chow diet (n=8, C1090-10, Altromine). Stereological parameters were obtained in diestrus cycle. The samples were processed through routine and standard paraffin embedding and were serially sectioned in 5-µm thickness then, every 10th section was saved, stained with Crossman's triple stain for counting and measuring. In all sampled sections mean follicle numbers, diameters, total ovarian volume cortex to medulla ratio (Vv), ovum to cell ratio in secondary follicle were examined in all sampled sections. The present results showed that weight of ovarian and amount of intraperitoneal adipose tissue and the body weight markedly increased in obese mice when compared with control groups. Moreover, follicle numbers (except primordial follicles) and diameters were significantly increased in obese mice. Cortex to medulla ratio (Vv) and ovum to cell ratio in secondary follicle were also considerably different between experimental and the control groups. The present findings indicate that obesity adversely affects overall ovarian morphology.

Could perturbed fetal development of the ovary contribute to the development of polycystic ovary syndrome in later life?

Polycystic ovary syndrome (PCOS) affects around 10% of young women, with adverse consequences on fertility and cardiometabolic outcomes. PCOS appears to result from a genetic predisposition interacting with developmental events during fetal or perinatal life. We hypothesised that PCOS candidate genes might be expressed in the fetal ovary when the stroma develops; mechanistically linking the genetics, fetal origins and adult ovarian phenotype of PCOS. In bovine fetal ovaries (n = 37) of 18 PCOS candidate genes only SUMO1P1 was not expressed. Three patterns of expression were observed: early gestation (FBN3, GATA4, HMGA2, TOX3, DENND1A, LHCGR and FSHB), late gestation (INSR, FSHR, and LHCGR) and throughout gestation (THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1). A splice variant of FSHB exon 3 was also detected early in the bovine ovaries, but exon 2 was not detected. Three other genes, likely to be related to the PCOS aetiology (AMH, AR and TGFB1I1), were also expressed late in gestation. Significantly within each of the three gene groups, the mRNA levels of many genes were highly correlated with each other, despite, in some instances, being expressed in different cell types. TGFβ is a well-known stimulator of stromal cell replication and collagen synthesis and TGFβ treatment of cultured fetal ovarian stromal cells inhibited the expression of INSR, AR, C8H9orf3 and RAD50 and stimulated the expression of TGFB1I1. In human ovaries (n = 15, < 150 days gestation) many of the same genes as in bovine (FBN3, GATA4, HMGA2, FSHR, DENND1A and LHCGR but not TOX3 or FSHB) were expressed and correlated with each other. With so many relationships between PCOS candidate genes during development of the fetal ovary, including TGFβ and androgen signalling, we suggest that future studies should determine if perturbations of these genes in the fetal ovary can lead to PCOS in later life.